CD-R 1 contains the Sequence Listing formatted in plain ASCII text. CD-R 1 is labeled with Identification No. PL-0017 US, 1 of 3, Copy 1. The file containing the Sequence Listing is entitled p10017.txt, created on Nov. 22, 2000, and is 3,929 KB in size.
CD-R 2 is an exact copy of CD-R 1. CD-R 2 is labeled with Identification No. PL-0017 US, 2 of 3, Copy 2.
CD-R 3 contains the Computer Readable Form of the Sequence Listing in compliance with 37 C.F.R. xc2xa71.821(e), and specified by 37 C.F.R. xc2xa71.824. CD-R 3 is labeled with Identification No. PL-0017 US, 3 of 3, Copy 3. The file containing the Sequence Listing is entitled pl0017.txt, created on Nov. 22, 2000, and is 3,929 KB in size.
The disclosure of the Sequence Listing submitted as an electronic document on compact disc as described above are to be part of the permanent USPTO record of this patent application and are hereby expressly incorporated by reference.
The present invention relates to nucleic and amino acid sequences derived from corn ear and to the use of these sequences in the identification, evaluation, and alteration of desired characteristics associated with growth and development, disease resistance, environmental adaptability, quality, and yield.
The field of plant breeding deals with the manipulation of plant genomes with the purpose of improving characteristics of the plant. Plant breeders use data and methodology from plant physiology, genetics, biochemistry, pathology, statistics, and molecular biology. One of the most improved hybrid crops is corn, Zea mays (L.). Corn is presently the second-most economically important crop in the United States. Acreage of field corn (used for livestock feed, corn starch, corn syrup, fuel ethanol, and oil) and sweet corn (used fresh or processed for human consumption) exceeds that of any other agronomic crop. Annual losses, reduction in quality and yield, due to diseases and infestation may range from 7 to 17%. Studies of corn may be used as a model for other economically important agronomic grasses.
Corn is a monocotyledonous plant which has one seed leaf, uses the C4 photosynthetic pathway, and has scattered vascular bundles. The mature plant is made up of roots, stem, leaves, and reproductive structures. The root system functions to anchor the plant and to absorb water and nutrients. The corn stem consists of a series of nodes, each bearing one bladelike leaf with parallel veins, and internodes, elongated stem sections. The stem and its leaves are commonly referred to as a stalk. Leaves arise alternately and are arranged in two rows along either side of the stem. The male reproductive structure is the tassel, and its flowers produce pollen. The female reproductive structure is the ear, and its flowers each have a silk for pollination. When a pollen grain is shed onto a silk and germinates, a pollen tube grows down the silk and delivers two sperm to the female gametophyte. Within the gametophyte, one sperm fertilizes the egg, and the other, two polar nuclei. The embryo and endosperm produced by this double fertilization develop into the seed which matures in about two months.
The vegetative (V) and reproductive (R) stages of growth for a corn plant are as follows: VE-emergence from the soil of the seedling leaf; V1-first true leaf; V2-second leaf; . . . V(n)-nth leaf; VT-tasseling stage; and reproductive stages, R1-silking; R2-blister; R3-milk; R4-dough; R5-dent; and R6-physiological maturity (Ritchie, S. W. et al. (1986) How a Corn Plant Develops, Iowa State University Cooperative Extension Service, Ames IA 48:1-21).
Corn Ear
Ear shoots are initiated at V3. As the primary apical meristem makes the transition from vegetative growth to tassel formation, the plant also begins to initiate buds in the axils of vegetative leaves. Ear primordia arise beginning at about the sixth node and proceed upward until about two nodes below the developing tassel. The earliest anatomical indications of ear development are periclinal divisions in the second and third cell layers of the shoot meristem followed by divisions in the outermost layer. This axillary floral meristem becomes organized and elongates to produce 8-14 husks which surround the developing cob. The husks remain shorter, and the sheaths become thinner and broader than those of ordinary leaves. Although the outer husks are arranged in two rows, the inner ones can be in several rows. During cob development, the floral meristem elongates and bifurcates one or more times; each bifurcation produces bracts in two ranks and eventually determines the number of rows of kernels on the cob. Each bract produces two spikelets, and each spikelet ultimately produces a single floret. Spikelet development parallels that of other graminaceous flowers and produces the following succession of flower parts (from the outside to inside): glumes, lemma, palea, stamen primordia which abort, and a terminal gynoecium. Gyonecial development begins with the enlargement of the ovary wall around the ovule followed by terminal elongation to form the silk (Sundberg, M. D., et al. (1995) Amer J Bot 82:64-74; Cheng, P. C., et al. (1983) Amer J Bot 70:450-462; and Johri, M. M. and Coe, Jr., E. H. (1983) Dev Biol 97:154-172).
Ear shoots are often visible at V6, and by V9, many ear shoots can be seen with appropriate dissection. An ear shoot may develop at every above-ground node with the exception of the last six to eight nodes below the tassel. Consistent with the age of each node and determined apical development, each ear develops faster than the ear originating above it on the stalk. However, growth of most lower ears eventually slows, and only the upper one or two ears ever mature and are harvested. At V12, the number of potential kernels on each ear and the size of the ear are determined. Although the number of rows of kernels per ear has already been established, cob elongation and the determination of the number of kernels per row will not be complete until about one week before silking, about V17.
By V15, upper ear development has surpassed that of lower ears, and silks are just beginning to grow from the ovules. Between V18 and R1, the silks grow acropetally from the basal ovules to those at the ear tip. R1 begins when any silks are visible outside the husks. Pollination occurs when wind borne pollen grains fall on these new, moist silks. Germination and growth of the pollen grain down the silk to the ovule where fertilization occurs takes about 24 hours. Generally two to three days are required for all silks on a single ear to be exposed and pollinated. The silks continue to elongate until they are fertilized or environmental or biological conditions cause their demise. The fertilized ovules immediately begin kernel development (Ritchie, S. W. et al. (1986) How a Corn Plant Develops, Iowa State University Cooperative Extension Service, Ames IA 48:1-21).
Problems of Corn Ears
All parts of the corn plant are susceptible to diseases, insect infestations, and stress. These conditions are usually diagnosed by their above-ground leaf, stalk, fruit and/or seed symptoms and are caused by fungi, bacteria, mycoplasmas and related organisms, viruses, nematodes, parasitic seed plants, insects and mites, and abnormal environmental conditions.
Fungal diseases are spread by spores that germinate under favorable conditions of temperature and moisture. Spores germinate to produce branched threads called hyphae that infect plants by direct penetration of the epidermis or through natural openings or wounds. Resting bodies (chlamydospores, sclerotia) allow fungi to survive under unfavorable conditions for long periods in the soil or in plant debris.
Head smut, caused by the fungus, Sphacelotheca reiliana, occurs in the deltas and mountain valleys of the Pacific Coast states, in the high plains of Texas, and in southeastern Europe. This soil-borne fungus appears in fields where nitrogen is deficient at the time of ear and tassel formation. Galls containing masses of brownish-black spores replace tassels and ears. When infection of the tassel is confined to individual spikelets, it can result in shoot-like growth on the tassel. Smutted ears are small and either are entirely replaced by a black powdery mass of spores or are aborted with shoot-like growth replacing the normal ear. Although tassels may appear healthy when smut forms in the ears, the plants do not produce pollen. Head smut galls form around heavy strands of vascular tissue which remain as the gall disintegrates. Infected plants are stunted and produce no grain. The fungal spores are dispersed by wind and persist in the soil where they infect germinating seedlings. In such situations, the mycelium systematically infects the young plant and invades the reproductive tissues. Other fungal diseases of ears include common and false smuts, and Gibberella and gray ear rots.
Plant-pathogenic bacteria are unicellular non-spore-forming rods with or without flagella which are spread by cultural practices, animals, water, and soil. Bacteria enter plants through wounds or natural openings and multiply rapidly inside the plant where they cause death of cells; abnormal growths; block water-conducting tissue; or break down the tissue structure. They can remain dormant on or within plant tissue, insects, soil, or equipment.
Stewart""s bacterial wilt caused by Erwinia stewartii, is common in eastern North America and has been reported in Europe. In susceptible corn varieties, leaves die prematurely, yield is reduced, and the plant becomes more susceptible to stalk rots. The bacteria can spread to all parts of the plant including the kernels. E. stewartii is identified in the field by plant symptoms and the yellow ooze which comes from cut ends of infected stalks or leaves. E. stewartii is spread by beetles; however, only the corn flea beetle (Chaetocnema pulicaria) overwinters and spreads the bacteria the next growing season. Mineral nutrition also influences susceptibility to infection: nitrogen in the form of ammonium and phosphorus increase susceptibility, and high calcium and potassium levels decrease susceptibility. Disease severity may also be aggravated by high temperatures.
Viruses are submicroscopic particles composed of nucleic acid and protein which are transmitted by biological vectors, e.g., aphids, leafhoppers, planthoppers, beetles, and other insects. A few corn viruses are seed-transmitted or overwinter in weed and crop plants or insect bodies. Viruses induce a variety of local or systematic symptoms (e.g., mosaic patterns, yellowing or reddening of the foliage, stunting, ringspots, and necrosis) which may be exacerbated by environmental conditions. Viruses which affect field and sweet corn include maize chlorotic dwarf; maize chlorotic mottle; maize dwarf mosaic; and corn lethal necrosis (a complex of maize chlorotic mottle virus and maize dwarf mosaic).
Maize Dwarf Mosaic Virus (MDMV) is a long, flexuous, rod-shaped virus. It is transmitted on seed or by over 20 species of aphids which acquire the virus from other grasses and transmit it to corn. Plants may be slightly stunted, and reduction in ear size and seed set may occur. MDMV symptoms are highly variable and include narrow, light green or yellowish streaks along the leaf veins and/or dark green xe2x80x9cislandsxe2x80x9d on a yellow background. Symptoms may be present on all leaves and husks that develop following infection. Both environmental conditions and the developmental stage at which the plant is infected may cause a reduction in yield; infection of young plants early in the season causes the most significant losses. In addition, early infection may predispose corn to root and stalk rots and premature death.
Corn lethal necrosis, found in Kansas and Nebraska, results from the synergistic interaction of maize chlorotic mottle virus (MCMV) in combination with either MDMV strain-A or -B or wheat streak mosaic virus (WSMV). Yield loss greatly exceeds that predicted from the cumulative effects of the individual viruses and may approach 100%. Symptoms include severe mottling, browning of the tassel and leaves, and early death of the plant. Late infections cause ears to fill incompletely and husks to dry prematurely. Potential vectors of these viruses may be: corn rootworm beetles (Diabrotica spp.) for MCMV, greenbug (Schizaphi graminum) and aphids (Rhopalosiphum maidis) for MDMV, and wheat curl mite (Eriophyes tulipae) for WSMV.
Important pests of corn include European corn, southwestern corn, and common stalk borers; corn rootworm; chinch bug; corn ear worm; armyworms; grasshoppers; corn leaf aphid; corn flea, dusky sap, and Japanese beetles; and spider mites. The European corn borer (Ostrinia nubilalis), introduced into the U.S. from Europe in 1909, has spread throughout the United States and into Canada. The European corn borer infests over 200 plants, but corn is a preferred host. The borer""s yearly life cycle varies from one generation in the northern areas to three in the south. The adult female is pale yellow brown with irregular darker bands on her wings; the male is darker with olive brown wings. The larva has flesh-color body with small, round, brown spots and a black head. Before pupating, it is about 25 mm. The early instars tunnel all parts of stalks and ears.
In corn, the larvae first feed in the whorl then bore down midribs of leaves into the stalk leaving frass and silk near their entrance holes. Borers weaken stalks and interfere with the movement of plant nutrients thus reducing yields. In infested corn plants, ears develop poorly or drop, and tassels and stalks may break.
Environmental conditions also affect growth, development, and yield by altering pathogen activity or host physiology. The severity of the excess, deficiency, or imbalance of soil nutrients or water; extreme soil acidity or alkalinity; very high or low temperatures; air pollutants; or mechanical, pesticide, or other injury varies with the stage of plant maturity during which the disturbance occurs and the plant organ involved. Phosphorus deficiency in young corn plants causes small ears which are often twisted with irregular kernel rows and imperfectly developed ear tips. Potassium deficiency also causes ears to be small, chaffy, and dull with pointed, poorly developed tips. (Shurtleff, M. C. et al. (1980) Compendium of Corn Diseases, APS Press, St. Paul Minn., 105 pp.).
Corn Disease Control
To control corn diseases, it is necessary to disrupt disease development. Intervention requires understanding the pathogens, the spread of pathogens, disease cycles, and plant resistance. Disease control may be achieved by a single procedure (chemical sprays) or by the integrated use of environmental, genetic, and chemical factors. Successful, long-term disease control generally includes planting disease-resistant varieties, applying chemical control, and implementing sanitary cultural methods.
The present invention provides nucleic acid sequences comprising corn ear-derived polynucleotides (cdps) as presented in the Sequence Listing. Some of the cdps uniquely identify structural, functional, and regulatory genes of the corn ear. The invention encompasses oligonucleotides, fragments, and derivatives of the cdps and provides nucleic acid sequences complementary to the nucleic acid sequences listed in the Sequence Listing.
The present invention further provides the following cdps of particular interest as identified by SEQ ID NO: 3250 (700611189H1), SEQ ID NO: 3392 (700611501H1), SEQ ID NO: 2031 (700551933H1), SEQ ID NO: 1954 (700551804H1), SEQ ID NO: 2582 (700552928H1), SEQ ID NO: 2016 (700551910H1), SEQ ID NO: 2311 (700552464H1), SEQ ID NO: 2238 (700552317H1), SEQ ID NO: 2096 (700552062H1), SEQ ID NO: 2152 (700552161H1), SEQ ID NO: 636 (700549563H1), SEQ ID NO: 1359 (700550807H1), SEQ ID NO: 811 (700549876H1), SEQ ID NO: 1817 (700551570H1), SEQ ID NO: 2648 (700553058H1), SEQ ID NO: 1460 (700550984H1), SEQ ID NO: 2983 (700282115H1), SEQ ID NO: 2061 (700551985H1), SEQ ID NO: 3095 (700610867H1), SEQ ID NO: 100 (700548530H1), SEQ ID NO: 2 (700548303H1), SEQ ID NO: 3307 (700611293H1), SEQ ID NO: 2160 (700552173H1), SEQ ID NO: 2104 (700552073H1), SEQ ID NO: 2058 (700551981H1), SEQ ID NO: 2177 (700552202H1), SEQ ID NO: 1190 (700550523H1), SEQ ID NO: 2665 (700553084H1), SEQ ID NO: 2510 (700552784H1), SEQ ID NO: 1406 (700550886H1), SEQ ID NO: 1196 (700550530H1), SEQ ID NO: 1127 (700550431H1), SEQ ID NO: 271 (700548896H1), SEQ ID NO: 1126 (700550430H1), SEQ ID NO: 738 (700549740H1), SEQ ID NO: 548 (700549421H1), SEQ ID NO: 542 (700549412H1), SEQ ID NO: 547 (700549420H1), SEQ ID NO: 2114 (700552089H1), SEQ ID NO: 3321 (700611324H1), SEQ ID NO: 2416 (700552643H1), SEQ ID NO: 2430 (700552661H1), SEQ ID NO: 830 (700549908H1), SEQ ID NO: 2182 (700552213H1), SEQ ID NO: 1135 (700550444H1), SEQ ID NO: 1742 (700551432H1), SEQ ID NO: 1633 (700551252H1), SEQ ID NO: 880 (700549994H1), SEQ ID NO: 2234 (700552312H1), SEQ ID NO: 1608 (700551216H1), SEQ ID NO: 2985 (700282117H1), SEQ ID NO: 234 (700548810H1), SEQ ID NO: 1273 (700550651H1), SEQ ID NO: 349 (700549051H1), SEQ ID NO: 3553 (700611877H1), SEQ ID NO: 364 (700549083H1), SEQ ID NO: 2014 (700551904H1), SEQ ID NO: 2962 (700282075H1), SEQ ID NO: 2390 (700552590H1), SEQ ID NO: 1814 (700551565H1), SEQ ID NO: 2892 (700553450H1), SEQ ID NO: 959 (700550142H1), SEQ ID NO: 3069 (700610808H1), SEQ ID NO: 3543 (700611844H1), SEQ ID NO: 3414 (700611544H1), SEQ ID NO: 1097 (700550384H1), SEQ ID NO: 1197 (700550531H1), SEQ ID NO: 1230 (700550584H1), SEQ ID NO: 1648 (700551276H1), SEQ ID NO: 2077 (700552025H1), SEQ ID NO: 3637 (700612029H1), SEQ ID NO: 2429 (700552659H1), SEQ ID NO: 2308 (700552459H1), SEQ ID NO: 2402 (700552620H1), SEQ ID NO: 1278 (700550659H1), SEQ ID NO: 1279 (700550660H1), SEQ ID NO: 1083 (700550360H1), SEQ ID NO: 485 (700549323H1), SEQ ID NO: 645 (700549585H1), SEQ ID NO: 222 (700548774H1), SEQ ID NO: 344 (700549039H1), SEQ ID NO: 879 (700549992H1), SEQ ID NO: 697 (700549667H1), SEQ ID NO: 1257 (700550630H1), SEQ ID NO: 902 (700550036H1), SEQ ID NO: 956 (700550134H1), SEQ ID NO: 67 (700548448H1), SEQ ID NO: 32 (700548366H1), SEQ ID NO: 144 (700548623H1), SEQ ID NO: 1074 (700550338H1), SEQ ID NO: 1201 (700550538H1), SEQ ID NO: 339 (700549027H1), SEQ ID NO: 2115 (700552091H1), SEQ ID NO: 2171 (700552190H1), 40 SEQ ID NO: 2172 (700552191H1), SEQ ID NO: 3108 (700610893H1), SEQ ID NO: 3160 (700610993H1), SEQ ID NO: 3207 (700611093H1), SEQ ID NO: 239 (700548820H1), SEQ ID NO: 2247 (700552332H1), SEQ ID NO: 279 (700548915H1), SEQ ID NO: 3806 (700612280H1), SEQ ID NO: 3732 (700612174H1), SEQ ID NO: 3814 (700612292H1), SEQ ID NO: 2158 (700552171H1), SEQ ID NO: 1184 (700550516H1), SEQ ID NO: 1870 (700551656H1), SEQ ID NO: 2324 (700552483H1), SEQ ID NO: 2375 (700552569H1), SEQ ID NO: 2454 (700552691H1), SEQ ID NO: 1621 (700551231H1), SEQ ID NO: 3735 (700612180H1), SEQ ID NO: 3566 (700611907H1), SEQ ID NO: 264 (700548874H1), SEQ ID NO: 1025 (700550257H1), SEQ ID NO: 535 (700549401H1), SEQ ID NO: 951 (700550126H1), SEQ ID NO: 354 (700549060H1), SEQ ID NO: 332 (700549012H1), SEQ ID NO: 936 (700550089H1), SEQ ID NO: 781 (700549822H1), SEQ ID NO: 1326 (700550749H1), SEQ ID NO: 3564 (700611904H1), SEQ ID NO: 3565 (700611905H1), SEQ ID NO: 1444 (700550962H1), SEQ ID NO: 1994 (700551863H1), SEQ ID NO: 3781 (700612249H1), SEQ ID NO: 2221 (700552281H1), SEQ ID NO: 3313 (700611306H1), SEQ ID NO: 3213 (700611106H1), SEQ ID NO: 3354 (700611405H1), SEQ ID NO: 213 (700548752H1), SEQ ID NO: 99 (700548523H1), SEQ ID NO: 359 (700549072H1), SEQ ID NO: 356 (700549067H1), SEQ ID NO: 992 (700550195H1), SEQ ID NO: 1228 (700550580H1), SEQ ID NO: 3333 (700611357H1), SEQ ID NO: 3381 (700611465H1), SEQ ID NO: 3699 (700612123H1), SEQ ID NO: 3634 (700612024H1), SEQ ID NO: 1432 (700550935H1), SEQ ID NO: 1327 (700550750H1), SEQ ID NO: 1256 (700550629H1), SEQ ID NO: 1068 (700550330H1), SEQ ID NO: 744 (700549752H1), SEQ ID NO: 892 (700550022H1), SEQ ID NO: 2697 (700553128H1), SEQ ID NO: 1708 (700551374H1), SEQ ID NO: 3347 (700611392H1), SEQ ID NO: 3560 (700611894H1), SEQ ID NO: 2036 (700551941H1), SEQ ID NO: 2028 (700551929H1), SEQ ID NO: 3703 (700612129H1), SEQ ID NO: 3767 (700612229H1), SEQ ID NO: 1008 (700550233H1), SEQ ID NO: 997 (700550214H1), SEQ ID NO: 472 (700549301H1), SEQ ID NO: 477 (700549309H1), SEQ ID NO: 2290 (700552436H1), SEQ ID NO: 2313 (700552468H1), SEQ ID NO: 3172 (700611010H1), SEQ ID NO: 2450 (700552686H1), SEQ ID NO: 524 (700549380H1), SEQ ID NO: 1417 (700550911H1), SEQ ID NO: 1383 (700550848H1), SEQ ID NO: 619 (700549540H1), SEQ ID NO: 1551 (700551129H1), SEQ ID NO: 1577 (700551161H1), SEQ ID NO: 250 (700548841H1), SEQ ID NO: 146 (700548625H1), SEQ ID NO: 1711 (700551380H1), SEQ ID NO: 1765 (700551476H1), SEQ ID NO: 1720 (700551391H1), SEQ ID NO: 905 (700550041H1), SEQ ID NO: 3391 (700611496H1), SEQ ID NO: 3349 (700611396H1), SEQ ID NO: 2126 (700552124H1), SEQ ID NO: 2030 (700551932H1), SEQ ID NO: 998 (700550215H1), SEQ ID NO: 1526 (700551091H1), SEQ ID NO: 1756 (700551460H1), SEQ ID NO: 474 (700549303H1), SEQ ID NO: 1177 (700550507H1), SEQ ID NO: 939 (700550093H1), SEQ ID NO: 2848 (700553376H1), SEQ ID NO: 1794 (700551534H1), SEQ ID NO: 2460 (700552701H1), SEQ ID NO: 2621 (700553001H1), SEQ ID NO: 216 (700548758H1), SEQ ID NO: 1213 (700550558H1), SEQ ID NO: 1733 (700551417H1), SEQ ID NO: 1669 (700551309H1), SEQ ID NO: 19 (700548332H1), SEQ ID NO: 614 (700549532H1), SEQ ID NO: 748 (700549762H1), SEQ ID NO: 2644 (700553051H1), SEQ ID NO: 2876 (700553418H1), SEQ ID NO: 3490 (700611717H1), SEQ ID NO: 106 (700548542H1), SEQ ID NO: 94 (700548510H1), SEQ ID NO: 2767 (700553233H1), SEQ ID NO: 1854 (700551635H1), SEQ ID NO: 2033 (700551937H1), SEQ ID NO: 2034 (700551938H1), SEQ ID NO: 1360 (700550808H1), SEQ ID NO: 3280 (700611242H1), SEQ ID NO: 629 (700549550H1), SEQ ID NO: 678 (700549642H1), SEQ ID NO: 491 (700549330H1), SEQ ID NO: 1620 (700551230H1), SEQ ID NO: 1456 (700550980H1), SEQ ID NO: 1524 (700551088H1), SEQ ID NO: 2631 (700553025H1), SEQ ID NO: 2639 (700553042H1), SEQ ID NO: 3546 (700611857H1), SEQ ID NO: 3510 (700611757H1), SEQ ID NO: 1301 (700550707H1), SEQ ID NO: 1063 (700550324H1), SEQ ID NO: 2255 (700552366H1), SEQ ID NO: 1738 (700551424H1), SEQ ID NO: 3419 (700611551H1), SEQ ID NO: 3468 (700611651H1), SEQ ID NO: 112 (700548554H1), SEQ 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(700350427H1), SEQ ID NO: 4484 (700348505H1), SEQ ID NO: 4890 (700349220H1), SEQ ID NO: 4892 (700349224H1), SEQ ID NO: 5550 (700350330H1), SEQ ID NO: 6404 (700351740H1), SEQ ID NO: 4764 (700349002H1), SEQ ID NO: 7266 (700381438H1), SEQ ID NO: 4003 (700347516H1), SEQ ID NO: 4097 (700347694H1), SEQ ID NO: 5176 (700349729H1), SEQ ID NO: 4630 (700348755H1), SEQ ID NO: 6652 (700352168H1), SEQ ID NO: 4321 (700348229H1), SEQ ID NO: 6113 (700351254H1), SEQ ID NO: 5064 (700349532H1), SEQ ID NO: 5415 (700350106H1), SEQ ID NO: 6304 (700351573H1), SEQ ID NO: 6577 (700352043H1), SEQ ID NO: 7582 (700381971H1), SEQ ID NO: 3853 (700282260H2), SEQ ID NO: 6876 (700352570H1), SEQ ID NO: 6796 (700352419H1), SEQ ID NO: 4709 (700348907H1), SEQ ID NO: 5528 (700350303H1), SEQ ID NO: 6833 (700352475H1), SEQ ID NO: 5653 (700350481H1), SEQ ID NO: 6910 (700380827H1), SEQ ID NO: 3944 (700347413H1), SEQ ID NO: 6696 (700352248H1), SEQ ID NO: 7122 (700381160H1), SEQ ID NO: 7274 (700381453H1), SEQ ID NO: 7087 (700381106H1), SEQ ID NO: 4858 (700349153H1), SEQ ID NO: 4335 (700348255H1), SEQ ID NO: 5372 (700350040H1), SEQ ID NO: 4500 (700348526H1), SEQ ID NO: 5133 (700349636H1), SEQ ID NO: 5003 (700349411H1), SEQ ID NO: 3938 (700282394H2), SEQ ID NO: 7330 (700381549H1), SEQ ID NO: 6639 (700352150H1), SEQ ID NO: 4170 (700347868H1), SEQ ID NO: 4351 (700348279H1), SEQ ID NO: 4907 (700349243H1), SEQ ID NO: 7145 (700381201H1), SEQ ID NO: 5659 (700350491H1), SEQ ID NO: 4535 (700348590H1), SEQ ID NO: 6198 (700351390H1), SEQ ID NO: 6191 (700351374H1), SEQ ID NO: 5156 (700349691H1), SEQ ID NO: 6481 (700351876H1), SEQ ID NO: 4199 (700347944H1), SEQ ID NO: 4569 (700348646H1), SEQ ID NO: 7165 (700381227H1), SEQ ID NO: 4945 (700349322H1), SEQ ID NO: 4555 (700348623H1), SEQ ID NO: 4937 (700349308H1), SEQ ID NO: 4944 (700349320H1), SEQ ID NO: 4885 (700349205H1), SEQ ID NO: 5244 (700349839H1), SEQ ID NO: 6164 (700351339H1), SEQ ID NO: 4649 (700348787H1), SEQ ID NO: 4546 (700348611H1), SEQ ID NO: 6288 (700351544H1), SEQ ID NO: 5445 (700350153H1), SEQ ID NO: 3839 (700282240H1), SEQ ID NO: 4543 (700348607H1), SEQ ID NO: 5378 (700350051H1), SEQ ID NO: 4652 (700348795H1), SEQ ID NO: 5896 (700350862H1), SEQ ID NO: 7106 (700381129H1), SEQ ID NO: 4742 (700348966H1), SEQ ID NO: 5817 (700350731H1), SEQ ID NO: 6260 (700351502H1), SEQ ID NO: 4648 (700348785H1), SEQ ID NO: 4211 (700347970H1), SEQ ID NO: 6930 (700380858H1), SEQ ID NO: 4560 (700348628H1), SEQ ID NO: 4565 (700348638H1), SEQ ID NO: 6687 (700352237H1), SEQ ID NO: 4949 (700349329H1), SEQ ID NO: 5577 (700350369H1), SEQ ID NO: 6140 (700351304H1), SEQ ID NO: 6359 (700351660H1), SEQ ID NO: 5259 (700349861H1), SEQ ID NO: 7118 (700381149H1), SEQ ID NO: 3957 (700347434H1), SEQ ID NO: 4758 (700348990H1), SEQ ID NO: 4001 (700347514H1), SEQ ID NO: 6704 (700352261H1), SEQ ID NO: 6892 (700380802H1), SEQ ID NO: 5232 (700349813H1), SEQ ID NO: 7337 (700381559H1), SEQ ID NO: 4121 (700347749H1), SEQ ID NO: 4331 (700348249H1), SEQ ID NO: 6350 (700351645H1), SEQ ID NO: 5235 (700349820H1), SEQ ID NO: 5609 (700350414H1), SEQ ID NO: 6720 (700352288H1), SEQ ID NO: 7406 (700381678H1), SEQ ID NO: 5504 (700350265H1), SEQ ID NO: 4383 (700348334H1), SEQ ID NO: 4131 (700347763H1), SEQ ID NO: 4423 (700348396H1), SEQ ID NO: 7329 (700381547H1), SEQ ID NO: 4093 (700347688H1), SEQ ID NO: 4310 (700348209H1), SEQ ID NO: 6681 (700352229H1), SEQ ID NO: 5263 (700349865H1), SEQ ID NO: 6254 (700351489H1), SEQ ID NO: 6442 (700351811H1), SEQ ID NO: 5226 (700349807H1), SEQ ID NO: 5308 (700349935H1), SEQ ID NO: 6730 (700352306H1), SEQ ID NO: 7499 (700381835H1), SEQ ID NO: 5902 (700350873H1), SEQ ID NO: 7531 (700381889H1), SEQ ID NO: 4371 (700348314H1), SEQ ID NO: 7600 (7b0381994H1), SEQ ID NO: 6354 (700351650H1), SEQ ID NO: 4653 (700348801H1), SEQ ID NO: 3941 (700347405H1), SEQ ID NO: 4206 (700347960H1), SEQ ID NO: 6832 (700352474H1), SEQ ID NO: 4337 (700348259H1), SEQ ID NO: 4193 (700347929H1), SEQ ID NO: 6220 (700351433H1), SEQ ID NO: 5511 (700350274H1), SEQ ID NO: 6337 (700351625H1), SEQ ID NO: 6900 (700380811H1), SEQ ID NO: 7547 (700381923H1), SEQ ID NO: 5525 (700350293H1), SEQ ID NO: 4474 (700348485H1), SEQ ID NO: 6498 (700351910H1), SEQ ID NO: 4968 (700349359H1), SEQ ID NO: 6295 (700351554H1), SEQ ID NO: 7584 (700381973H1), SEQ ID NO: 4750 (700348978H1), SEQ ID NO: 5355 (700350018H1), SEQ ID NO: 4464 (700348467H1), SEQ ID NO: 6755 (700352345H1), SEQ ID NO: 4106 (700347718H1), SEQ ID NO: 4886 (700349211H1), SEQ ID NO: 5553 (700350334H1), SEQ ID NO: 4396 (700348356H1), SEQ ID NO: 5939 (700350936H1), SEQ ID NO: 6189 (70035137H1), SEQ ID NO: 5803 (700350709H1), SEQ ID NO: 4655 (700348803H1), SEQ ID NO: 6120 (700351265H1), SEQ ID NO: 4799 (700349054H1), SEQ ID NO: 5830 (700350748H1), SEQ ID NO: 6686 (700352236H1), SEQ ID NO: 4589 (700348678H1), SEQ ID NO: 5870 (700350819H1), SEQ ID NO: 5794 (700350692H1), SEQ ID NO: 4646 (700348782H1), SEQ ID NO: 4489 (700348513H1), SEQ ID NO: 4157 (700347830H1), SEQ ID NO: 5651 (700350478H1), SEQ ID NO: 5982 (700351019H1), SEQ ID NO: 4466 (700348472H1), SEQ ID NO: 4692 (700348872H1), SEQ ID NO: 6847 (700352506H1), SEQ ID NO: 4980 (700349375H1), SEQ ID NO: 4387 (700348343H1), SEQ ID NO: 4067 (700347631H1), SEQ ID NO: 5546 (700350324H1), SEQ ID NO: 3999 (700347509H1), SEQ ID NO: 4794 (700349046H1), SEQ ID NO: 4382 (700348333H1), SEQ ID NO: 5348 (700350008H1), SEQ ID NO: 4657 (700348806H1). These selected cdps represent unique, corn ear-specific polynucleotides which are used to produce a ear-specific profile of gene transcription, a transcript image.
The cdps are also used as a composition in methods to detect altered gene expression in inbred or hybrid plants. Such methods employ the cdps of the Sequence Listing, oligonucleotides, fragments, derivatives, or complementary sequences in hybridization technologies. The invention provides a method for detecting polynucleotides in a biological sample, the method comprising the steps of hybridizing a cdp to at least one of the nucleic acids in the biological sample, thereby forming a hybridization complex, and detecting the hybridization complex, wherein the presence of the complex correlates with the presence of the polynucleotide in the sample. An additional method provides for amplification of the nucleic acids of the biological sample prior to hybridization. The invention provides a method of screening a plurality of molecules for specific binding to a polynucleotide, the method comprising the steps of providing the plurality of molecules; combining the polynucleotide with each of the plurality of molecules for a time sufficient to allow binding under suitable conditions; and detecting binding of the polynucleotide to each of the plurality of molecules, thereby identifying the molecules which specifically bind the polynucleotide.
The invention further provides a method for recovering a regulatory element, the method comprising the steps of designing oligomers to at least one of the cdps, combining the oligomers with a DNA library under appropriate conditions to amplify the cdp, comparing the cdp with the amplified sequence to identify overlapping areas, identifying additional sequence beyond the overlapping areas, and repeating steps a) through d) until the regulatory element is recovered. The regulatory element is a ear-specific regulatory element which may be placed in an expression vector which is transformed into a corn plant. The regulatory element is of value in regulating the expression of introduced cdps.
The invention provides a purified corn ear-derived polynucleotide capable of expressing a corn ear-derived polypeptide. In one embodiment, the corn ear-derived polynucleotide is contained within an expression vector. In a second embodiment, the expression vector is contained within a host cell. The invention also provides a method for producing a corn ear-derived polypeptide, said method comprising the steps of culturing the host cells containing the ear-derived polynucleotide under conditions suitable for the expression of a corn ear-derived polypeptide, and recovering the corn ear-derived polypeptide from the cell culture.
The invention provides a purified corn ear-derived polypeptide (CDP) encoded by at least one of the cdps of the Sequence Listing. The invention also provides an anti-CDP antibody specific for a purified polypeptide encoded by the cdp. Such antibodies may be used for diagnostic purposes for the detection of CDPs in specific plant cells.
The invention further provides a method for identifying a test compound which specifically binds the CDP, the method comprising the steps of providing a test compound, combining the CDP with the test compound under suitable conditions for a time sufficient to allow binding, and detecting CDP binding to the test compound.
A portion of the disclosure of this patent document contains material which is subject to copyright protection. The copyright owner has no objection to the facsimile reproduction by anyone of the patent document or the patent disclosure, as it appears in the Patent and Trademark Office patent file or records, but otherwise reserves all copyright rights whatsoever.
The Sequence Listing is a compilation of nucleic acid sequences obtained by sequencing clone inserts or isolates of two corn ear cDNA libraries. Each sequence is identified by a sequence identification number (SEQ ID NO) and by an Incyte Clone number. TABLES 1 and 2 are compilations of Incyte Clones arranged as described below.
TABLE 1 lists homologous isolates from the two corn ear cDNA libraries prepared as described in the Examples. The first column contains Incyte Clone numbers. The Incyte Clone number provides a cross reference to the Sequence Listing. The second column contains a relevant GenBank identification number. The third and fourth columns represent product and log-likelihood scores (Karlin, S. and S. F. Altschul (1993) Proc. Nat. Acad. Sci. 90:5873-5877). The fifth column refers to the particular database and release of GenBank against which the Incyte Clone was searched and in which a related sequence was found. The sequences of this invention were compared to sequences in the GenBank plant, eukaryotic and protein databases, and most isolates list homology to sequences in those databases. The last column contains a description of the referenced GenBank sequence.
TABLE 2 is a compilation representing corn ear-specific gene activity as illustrated by sets of clustered or related sequences. Each cluster disclosed in the table contains unique or homologous sequences that are specific to the two corn ear cDNA libraries. The clones in a cluster may contain overlapping sequences or they may be related to or overlap a common reference sequence. Clusters are compiled by naming, matching, and counting all copies of the cdp. The minimum number of clones required to define a cluster is two; clusters containing two clones are found at the bottom of the table. Some clusters are characterized further by the homology of one or more sequences to a reference sequence which has a GenBank identifier (g) and description. Homologous sequences are more fully described in TABLE 1.
Before the nucleic acid sequences and methods are presented, it is to be understood that this invention is not limited to the particular methodologies, protocols, cell lines, vectors, and reagents described. Although particular embodiments are described, methods and materials similar or equivalent to these embodiments may be used to practice the invention. The preferred methods, devices, and materials set forth are not intended to limit the scope of the invention which is limited only by the appended claims.
The singular forms xe2x80x9caxe2x80x9d, xe2x80x9canxe2x80x9d, and xe2x80x9cthexe2x80x9d include plural reference unless the context clearly dictates otherwise. All technical and scientific terms have the meanings commonly understood by one of ordinary skill in the art. All publications are incorporated by reference for the purpose of describing and disclosing the cell lines, vectors, and methodologies which are presented and which might be used in connection with the invention. Nothing in the specification is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.
Definitions
As used herein, the lower case xe2x80x9ccdpxe2x80x9d refers to a nucleic acid sequence, while the upper case xe2x80x9cCDPxe2x80x9d refers to an amino acid sequence. A xe2x80x9cfull-lengthxe2x80x9d cdp refers to a nucleic acid sequence containing the entire coding region of a gene endogenously expressed in corn.
xe2x80x9cAdjuvantsxe2x80x9d are materials such as Freund""s, mineral gels (aluminum hydroxide), and surface active substances (lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin (KLH; Sigma-Aldrich, St. Louis Mo.), and dinitrophenol) which may be administered to increase a host""s immunological response.
xe2x80x9cAllelexe2x80x9d refers to an alternative form of a nucleic acid sequence. Alleles result from a xe2x80x9cmutationxe2x80x9d, and any given genome may have none, one, or many allelic forms. Mutations which give rise to alleles are ascribed to deletions, additions or substitutions of nucleotides. Each of these types of changes may occur alone, or in combination with the others, one or more times in a given nucleic acid sequence. The present invention encompasses allelic cdps.
xe2x80x9cAmino acid sequencexe2x80x9d refers to an oligopeptide, a peptide, a polypeptide, or a protein of either natural or synthetic origin. The amino acid sequence is not limited to the complete, endogenous amino acid sequence and may be a portion, epitope, variant, or derivative of a protein expressed by a corn nucleic acid sequence.
xe2x80x9cAmplificationxe2x80x9d refers to the production of additional copies of a sequence and is carried out using polymerase chain reaction (PCR) technologies well known in the art.
xe2x80x9cAntisensexe2x80x9d refers to nucleic acid sequences which are complementary to a specific DNA or RNA sequence. The antisense strand (negative or 3xe2x80x2-5xe2x80x2) is that nucleic acid strand that is complementary to the sense strand (positive or 5xe2x80x2-3xe2x80x2).
xe2x80x9cBiologically activexe2x80x9d refers to a peptide having a structural, regulatory biochemical, or immunological function of a naturally occurring peptide.
xe2x80x9cComplementaryxe2x80x9d refers to the natural association of nucleic acid sequences by base-pairing (A-G-T pairs with its complement T-C-A).
xe2x80x9cComplementaryxe2x80x9d between two single-stranded molecules may be partial or complete. The degree to which two sequences are complementary affects the efficiency of hybridization and amplification reactions.
xe2x80x9cDerivativexe2x80x9d refers to the chemical modification of a nucleic acid sequence by replacement of hydrogen by an alkyl, acyl, amino, or other group.
xe2x80x9cHomologyxe2x80x9d refers to sequence similarity either between a reference nucleic acid sequence and at least a fragment of a cdp or between a reference amino acid sequence and a portion of a CDP.
xe2x80x9cHybridizationxe2x80x9d refers to the process by which a strand of nucleic acid joins with a complementary strand through base pairing.
xe2x80x9cImmunogenicxe2x80x9d defines the capability of a natural, recombinant or synthetic oligopeptide, or polypeptide, to induce antibody production in appropriate animals or cells.
xe2x80x9cLabelingxe2x80x9d refers to the covalent or noncovalent joining of a polynucleotide, polypeptide or antibody with a reporter molecule that provides for a detectable and often measurable signal.
xe2x80x9cLinkersxe2x80x9d are short stretches of nucleotide sequence which may be added onto a vector or a cdp to create restriction endonuclease sites for ease in cloning. xe2x80x9cPolylinkersxe2x80x9d are engineered to include multiple restriction enzyme sites and provide for the use of both those enzymes which leave 5xe2x80x2 and 3xe2x80x2 overhangs (such as BamHI, EcoRI, and HindIII) or which provide a blunt end (such as EcoRV, SnaBI, and StuI).
xe2x80x9cNaturally occurringxe2x80x9d refers to an endogenous polynucleotide or polypeptide that may be isolated from viruses or prokaryotic or eukaryotic cells.
xe2x80x9cNucleic acid sequencexe2x80x9d refers to an oligomer, oligonucleotide, nucleotide or polynucleotide, and its fragments, and to DNA or RNA of genomic or synthetic origin which may be single- or double-stranded and represent the sense or complementary (antisense) strand.
xe2x80x9cOligomerxe2x80x9d refers to a nucleic acid sequence of at least about 10 nucleotides and as many as about 60 nucleotides, preferably about 15 to 30 nucleotides, that may be used as a primer or amplimer in amplification technologies. Oligomers are usually chemically synthesized.
xe2x80x9cPeptide nucleic acidxe2x80x9d (PNA) refers to an oligomer of at least six nucleotides to which an amino acid residue, such as lysine, and an amino group have been added. PNAs, also designated antigene agents, stop transcript elongation by binding to their complementary strand of nucleic acid.
xe2x80x9cPlant samplexe2x80x9d refers to a cell, chromosomes isolated from a cell, genomic DNA, RNA, or cDNA in solution or bound to a substrate; an extract from plant cells, a cleared tissue, a blot or imprint from the cut edge of a plant part, or the like.
A xe2x80x9cportionxe2x80x9d of an CDP may be selected based upon retention of biological or immunological characteristics shared with naturally occurring polypeptides derived from corn ear. For example, an antigenic portion of a CDP may be used to induce antibody in an appropriate host.
xe2x80x9cPost-translational modificationxe2x80x9d of a CDP may involve lipidation, glycosylation, phosphorylation, acetylation, racemization, proteolytic cleavage, and the like. These processes may occur synthetically or biochemically. Biochemical modifications will vary by cell type depending on the enzymatic milieu and the CDP.
xe2x80x9cProbexe2x80x9d refers to cdps or fragments thereof, which are used to detect identical, allelic or related nucleic acid sequences. xe2x80x9cPurifiedxe2x80x9d refers to molecules, either polynucleotides or polypeptides that are isolated or separated from their natural environment and are at least 60% free, preferably 75% free, and most preferably 90% free from other compounds with which they are naturally associated.
xe2x80x9cRegulatory elementxe2x80x9d refers to a nucleic acid sequence from nontranslated regions of a gene such as enhancers, promoters, introns, and 3xe2x80x2 untranslated regions which interact with host proteins to carry out transcription or translation.
xe2x80x9cReporterxe2x80x9d molecules are chemical or biochemical moieties used for labeling a nucleic acid, an amino acid, or an antibody. They include radionuclides; enzymes; fluorescent, chemiluminescent, or chromogenic agents; substrates; cofactors; inhibitors; magnetic particles; and the like.
xe2x80x9cSubstratexe2x80x9d refers to any suitable rigid or semi-rigid support including membranes, filters, chips, slides, wafers, fibers, magnetic or nonmagnetic beads, gels, capillaries or other tubing, plates, polymers, and microparticles. The substrate can have a variety of surface forms, such as wells, trenches, pins, channels and pores, to which the polynucleotides are bound.
xe2x80x9cTransformationxe2x80x9d refers to a process by which exogenous DNA enters and changes a recipient cell. It may occur under natural or artificial conditions using various methods well known in the art. Transformation may rely on any known method for the insertion of foreign nucleic acid sequences into a prokaryotic or eukaryotic host cell. The method is selected based on the host cell being transformed. Transformants include stably transformed cells in which the inserted DNA is capable of replication either as an autonomously replicating plasmid or as part of the host chromosome, as well as cells which transiently express the inserted DNA or RNA.
xe2x80x9cVariantxe2x80x9d refers to an amino acid sequence which differs from another sequence by at least one amino acid. The variant may have xe2x80x9cconservativexe2x80x9d changes (e.g. replacement of leucine with isoleucine), which does not affect structural or chemical properties; or more rarely, xe2x80x9cnonconservativexe2x80x9d changes (e.g. replacement of glycine with tryptophan), which may affect structural and/or chemical properties.
The Invention
In a particular embodiment, mRNA was isolated from corn ear and used to construct the SATMON022 and SATMON023 cDNA libraries. The invention relates to nucleic acid sequences comprising corn ear-derived polynucleotides (cdps) as presented in the Sequence Listing and to the use of these nucleic acid sequences. A xe2x80x9ccorn ear-derived polynucleotidexe2x80x9d refers to a cdp which may be naturally occurring, recombinant, synthetic, or semi-synthetic. A subset of clustered ear-specific cdps is given in TABLE 2. The cdps may be used to identify, isolate, or extend identical or related corn ear nucleic acid sequences from DNA libraries for the purpose of producing an entire coding region or recovering a regulatory element. The cdps may also be used in nucleic acid hybridization or amplification technologies to follow expression of desirable traits through plant breeding programs. The present invention provides for expression vectors and host cells containing nucleic acid sequences that encode CDPs or portions thereof and regulatory elements obtained using methods described herein. The CDPs may possess biological or immunological activity, or both. The invention provides for the use of purified CDPs to induce antibodies for diagnostic use and to identify test compounds which specifically bind the CDP.
Specifically, the present invention relates to the following subset of unique and ear-specific cdps whose transcripts occurred more than once in the two corn ear cDNA libraries. They are SEQ ID NO: 3250 (700611189H1), SEQ ID NO: 3392 (700611501H1), SEQ ID NO: 2031 (700551933H1), SEQ ID NO: 1954 (700551804H1), SEQ ID NO: 2582 (700552928H1), SEQ ID NO: 2016 (700551910H1), SEQ ID NO: 2311 (700552464H1), SEQ ID NO: 2238 (700552317H1), SEQ ID NO: 2096 (700552062H1), SEQ ID NO: 2152 (700552161H1), SEQ ID NO: 636 (700549563H1), SEQ ID NO: 1359 (700550807H1), SEQ ID NO: 811 (700549876H1), SEQ ID NO: 1817 (700551570H1), SEQ ID NO: 2648 (700553058H1), SEQ ID NO: 1460 (700550984H1), SEQ ID NO: 2983 (700282115H1), SEQ ID NO: 2061 (700551985H1), SEQ ID NO: 3095 (700610867H1), SEQ ID NO: 100 (700548530H1), SEQ ID NO: 2 (700548303H1), SEQ ID NO: 3307 (700611293H1), SEQ ID NO: 2160 (700552173H1), SEQ ID NO: 2104 (700552073H1), SEQ ID NO: 2058 (700551981H1), SEQ ID NO: 2177 (700552202H1), SEQ ID NO: 1190 (700550523H1), SEQ ID NO: 2665 (700553084H1), SEQ ID NO: 2510 (700552784H1), SEQ ID NO: 1406 (700550886H1), SEQ ID NO: 1196 (700550530H1), SEQ ID NO: 1127 (700550431H1), SEQ ID NO: 271 (700548896H1), SEQ ID NO: 1126 (700550430H1), SEQ ID NO: 738 (700549740H1), SEQ ID NO: 548 (700549421H1), SEQ ID NO: 542 (700549412H1), SEQ ID NO: 547 (700549420H1), SEQ ID NO: 2114 (700552089H1), SEQ ID NO: 3321 (700611324H1), SEQ ID NO: 2416 (700552643H1), SEQ ID NO: 2430 (700552661H1), SEQ ID NO: 830 (700549908H1), SEQ ID NO: 2182 (700552213H1), SEQ ID NO: 1135 (700550444H1), SEQ ID NO: 1742 (700551432H1), SEQ ID NO: 1633 (700551252H1), SEQ ID NO: 880 (700549994H1), SEQ ID NO: 2234 (700552312H1), SEQ ID NO: 1608 (700551216H1), SEQ ID NO: 2985 (700282117H1), SEQ ID NO: 234 (700548810H1), SEQ ID NO: 1273 (700550651H1), SEQ ID NO: 349 (700549051H1), SEQ ID NO: 3553 (700611877H1), SEQ ID NO: 364 (700549083H1), SEQ ID NO: 2014 (700551904H1), SEQ ID NO: 2962 (700282075H1), SEQ ID NO: 2390 (700552590H1), SEQ ID NO: 1814 (700551565H1), SEQ ID NO: 2892 (700553450H1), SEQ ID NO: 959 (700550142H1), SEQ ID NO: 3069 (700610808H1), SEQ ID NO: 3543 (700611844H1), SEQ ID NO: 3414 (700611544H1), SEQ ID NO: 1097 (700550384H1), SEQ ID NO: 1197 (700550531H1), SEQ ID NO: 1230 (700550584H1), SEQ ID NO: 1648 (700551276H1), SEQ ID NO: 2077 (700552025H1), SEQ ID NO: 3637 (700612029H1), SEQ ID NO: 2429 (700552659H1), SEQ ID NO: 2308 (700552459H1), SEQ ID NO: 2402 (700552620H1), SEQ ID NO: 1278 (700550659H1), SEQ ID NO: 1279 (700550660H1), SEQ ID NO: 1083 (700550360H1), SEQ ID NO: 485 (700549323H1), SEQ ID NO: 645 (700549585H1), SEQ ID NO: 222 (700548774H1), SEQ ID NO: 344 (700549039H1), SEQ ID NO: 879 (700549992H1), SEQ ID NO: 697 (700549667H1), SEQ ID NO: 1257 (700550630H1), SEQ ID NO: 902 (700550036H1), SEQ ID NO: 956 (700550134H1), SEQ ID NO: 67 (700548448H1), SEQ ID NO: 32 (700548366H1), SEQ ID NO: 144 (700548623H1), SEQ ID NO: 1074 (700550338H1), SEQ ID NO: 1201 (700550538H1), SEQ ID NO: 339 (700549027H1), SEQ ID NO: 2115 (700552091H1), SEQ ID NO: 2171 (700552190H1), SEQ ID NO: 2172 (700552191H1), SEQ ID NO: 3108 (700610893H1), SEQ ID NO: 3160 (700610993H1), SEQ ID 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(700381151H1), SEQ ID NO: 4430 (700348418H1), SEQ ID NO: 7215 (700381331H1), SEQ ID NO: 4473 (700348481H1), SEQ ID NO: 7082 (700381096H1), SEQ ID NO: 5600 (700350402H1), SEQ ID NO: 5271 (700349875H1), SEQ ID NO: 6505 (700351918H1), SEQ ID NO: 6731 (700352307H1), SEQ ID NO: 4836 (700349114H1), SEQ ID NO: 4665 (700348817H1), SEQ ID NO: 7210 (700381325H1), SEQ ID NO: 5970 (700350990H1), SEQ ID NO: 4835 (700349113H1), SEQ ID NO: 5524 (700350292H1), SEQ ID NO: 5482 (700350231H1), SEQ ID NO: 4591 (700348683H1), SEQ ID NO: 5490 (700350244H1), SEQ ID NO: 5883 (700350843H1), SEQ ID NO: 4399 (700348360H1), SEQ ID NO: 5068 (700349538H1), SEQ ID NO: 4116 (700347737H1), SEQ ID NO: 5074 (700349547H1), SEQ ID NO: 4590 (700348682H1), SEQ ID NO: 6023 (700351094H1), SEQ ID NO: 7528 (700381886H1), SEQ ID NO: 7071 (700381079H1), SEQ ID NO: 6316 (700351590H1), SEQ ID NO: 6424 (700351780H1), SEQ ID NO: 5862 (700350801H1), SEQ ID NO: 6369 (700351676H1), SEQ ID NO: 5411 (700350101H1), SEQ ID NO: 6850 (700352516H1), SEQ ID NO: 6546 (700351988H1), SEQ ID NO: 7562 (700381943H1), SEQ ID NO: 6211 (700351417H1), SEQ ID NO: 6659 (700352179H1), SEQ ID NO: 6950 (700380888H1), SEQ ID NO: 4047 (700347589H1), SEQ ID NO: 4934 (700349303H1), SEQ ID NO: 6137 (700351296H1), SEQ ID NO: 7372 (700381626H1), SEQ ID NO: 5192 (700349751H1), SEQ ID NO: 6089 (700351218H1), SEQ ID NO: 5515 (700350279H1), SEQ ID NO: 6795 (700352418H1), SEQ ID NO: 5638 (700350457H1), SEQ ID NO: 6192 (700351380H1), SEQ ID NO: 6199 (700351396H1), SEQ ID NO: 5773 (700350657H1), SEQ ID NO: 7141 (700381192H1), SEQ ID NO: 6748 (700352335H1), SEQ ID NO: 4070 (700347635H1), SEQ ID NO: 7447 (700381743H1), SEQ ID NO: 5802 (700350708H1), SEQ ID NO: 6509 (700351926H1), SEQ ID NO: 5131 (700349632H1), SEQ ID NO: 7116 (700381144H1), SEQ ID NO: 6820 (700352453H1), SEQ ID NO: 7515 (700381865H1), SEQ ID NO: 6911 (700380828H1), SEQ ID NO: 6718 (700352284H1), SEQ ID NO: 4557 (700348625H1), SEQ ID NO: 6862 (700352545H1), SEQ ID NO: 5856 (700350789H1), SEQ ID NO: 6982 (700380949H1), SEQ ID NO: 5556 (700350341H1), SEQ ID NO: 4905 (700349240H1), SEQ ID NO: 6124 (700351269H1), SEQ ID NO: 5816 (700350730H1), SEQ ID NO: 5823 (700350738H1), SEQ ID NO: 5680 (700350524H1), SEQ ID NO: 5388 (700350066H1), SEQ ID NO: 6669 (700352201H1), SEQ ID NO: 5703 (700350553H1), SEQ ID NO: 4103 (700347712H1), SEQ ID NO: 3943 (700347412H1), SEQ ID NO: 7252 (700381415H1), SEQ ID NO: 6019 (700351084H1), SEQ ID NO: 4690 (700348868H1), SEQ ID NO: 4718 (700348921H1), SEQ ID NO: 5320 (700349953H1), SEQ ID NO: 7105 (700381128H1), SEQ ID NO: 5742 (700350611H1), SEQ ID NO: 5923 (700350910H1), SEQ ID NO: 6004 (700351058H1), SEQ ID NO: 6574 (700352039H1), SEQ ID NO: 6600 (700352077H1), SEQ ID NO: 5167 (700349712H1), SEQ ID NO: 6356 (700351655H1), SEQ ID NO: 4672 (700348831H1), SEQ ID NO: 4394 (700348354H1), SEQ ID NO: 5660 (700350492H1), SEQ ID NO: 6163 (700351338H1), SEQ ID NO: 5534 (700350311H1), SEQ ID NO: 6738 (700352316H1), SEQ ID NO: 5725 (700350581H1), SEQ ID NO: 4760 (700348993H1), SEQ ID NO: 7480 (700381806H1), SEQ ID NO: 7288 (700381486H1), SEQ ID NO: 6516 (700351934H1), SEQ ID NO: 6512 (700351930H1), SEQ ID NO: 4136 (700347771H1), SEQ ID NO: 4115 (700347736H1), SEQ ID NO: 5477 (700350220H1), SEQ ID NO: 6522 (700351954H1), SEQ ID NO: 4158 (700347835H1), SEQ ID NO: 4619 (700348737H1), SEQ ID NO: 4676 (700348837H1), SEQ ID NO: 6420 (700351774H1), SEQ ID NO: 5181 (700349734H1), SEQ ID NO: 6403 (700351739H1), SEQ ID NO: 5549 (700350329H1), SEQ ID NO: 3834 (700282233H2), SEQ ID NO: 7132 (700381176H1), SEQ ID NO: 4420 (700348387H1), SEQ ID NO: 6343 (700351632H1), SEQ ID NO: 6105 (700351241H1), SEQ ID NO: 6064 (700351163H1), SEQ ID NO: 5396 (700350077H1), SEQ ID NO: 5006 (700349420H1), SEQ ID NO: 7488 (700381818H1), SEQ ID NO: 7416 (700381688H1), SEQ ID NO: 5031 (700349468H1), SEQ ID NO: 6746 (700352333H1), SEQ ID NO: 5616 (700350427H1), SEQ ID NO: 4484 (700348505H1), SEQ ID NO: 4890 (700349220H1), SEQ ID NO: 4892 (700349224H1), SEQ ID NO: 5550 (700350330H1), SEQ ID NO: 6404 (700351740H1), SEQ ID NO: 4764 (700349002H1), SEQ ID NO: 7266 (700381438H1), SEQ ID NO: 4003 (700347516H1), SEQ ID NO: 4097 (700347694H1), SEQ ID NO: 5176 (700349729H1), SEQ ID NO: 4630 (700348755H1), SEQ ID NO: 6652 (700352168H1), SEQ ID NO: 4321 (700348229H1), SEQ ID NO: 6113 (700351254H1), SEQ ID NO: 5064 (700349532H1), SEQ ID NO: 5415 (700350106H1), SEQ ID NO: 6304 (700351573H1), SEQ ID NO: 6577 (700352043H1), SEQ ID NO: 7582 (700381971H1), SEQ ID NO: 3853 (700282260H2), SEQ ID NO: 6876 (700352570H1), SEQ ID NO: 6796 (700352419H1), SEQ ID NO: 4709 (700348907H1), SEQ ID NO: 5528 (700350303H1), SEQ ID NO: 6833 (700352475H1), SEQ ID NO: 5653 (700350481H1), SEQ ID NO: 6910 (700380827H1), SEQ ID NO: 3944 (700347413H1), SEQ ID NO: 6696 (700352248H1), SEQ ID NO: 7122 (700381160H1), SEQ ID NO: 7274 (700381453H1), SEQ ID NO: 7087 (700381106H1), SEQ ID NO: 4858 (700349153H1), SEQ ID NO: 4335 (700348255H1), SEQ ID NO: 5372 (700350040H1), SEQ ID NO: 4500 (700348526H1), SEQ ID NO: 5133 (700349636H1), SEQ ID NO: 5003 (700349411H1), SEQ ID NO: 3938 (700282394H2), SEQ ID NO: 7330 (700381549H1), SEQ ID NO: 6639 (700352150H1), SEQ ID NO: 4170 (700347868H1), SEQ ID NO: 4351 (700348279H1), SEQ ID NO: 4907 (700349243H1), SEQ ID NO: 7145 (700381201H1), SEQ ID NO: 5659 (700350491H1), SEQ ID NO: 4535 (700348590H1), SEQ ID NO: 6198 (700351390H1), SEQ ID NO: 6191 (700351374H1), SEQ ID NO: 5156 (700349691H1), SEQ ID NO: 6481 (700351876H1), SEQ ID NO: 4199 (700347944H1), SEQ ID NO: 4569 (700348646H1), SEQ ID NO: 7165 (700381227H1), SEQ ID NO: 4945 (700349322H1), SEQ ID NO: 4555 (700348623H1), SEQ ID NO: 4937 (700349308H1), SEQ ID NO: 4944 (700349320H1), SEQ ID NO: 4885 (700349205H1), SEQ ID NO: 5244 (700349839H1), SEQ ID NO: 6164 (700351339H1), SEQ ID NO: 4649 (700348787H1), SEQ ID NO: 4546 (700348611H1), SEQ ID NO: 6288 (700351544H1), SEQ ID NO: 5445 (700350153H1), SEQ ID NO: 3839 (700282240H1), SEQ ID NO: 4543 (700348607H1), SEQ ID NO: 5378 (700350051H1), SEQ ID NO: 4652 (700348795H1), SEQ ID NO: 5896 (700350862H1), SEQ ID NO: 7106 (700381129H1), SEQ ID NO: 4742 (700348966H1), SEQ ID NO: 5817 (700350731H1), SEQ ID NO: 6260 (700351502H1), SEQ ID NO: 4648 (700348785H1), SEQ ID NO: 4211 (700347970H1), SEQ ID NO: 6930 (700380858H1), SEQ ID NO: 4560 (700348628H1), SEQ ID NO: 4565 (700348638H1), SEQ ID NO: 6687 (700352237H1), SEQ ID NO: 4949 (700349329H1), SEQ ID NO: 5577 (700350369H1), SEQ ID NO: 6140 (700351304H1), SEQ ID NO: 6359 (700351660H1), SEQ ID NO: 5259 (700349861H1), SEQ ID NO: 7118 (700381149H1), SEQ ID NO: 3957 (700347434H1), SEQ ID NO: 4758 (700348990H1), SEQ ID NO: 4001 (700347514H1), SEQ ID NO: 6704 (700352261H1), SEQ ID NO: 6892 (700380802H1), SEQ ID NO: 5232 (700349813H1), SEQ ID NO: 7337 (700381559H1), SEQ ID NO: 4121 (700347749H1), SEQ ID NO: 4331 (700348249H1), SEQ ID NO: 6350 (700351645H1), SEQ ID NO: 5235 (700349820H1), SEQ ID NO: 5609 (700350414H1), SEQ ID NO: 6720 (700352288H1), SEQ ID NO: 7406 (700381678H1), SEQ ID NO: 5504 (700350265H1), SEQ ID NO: 4383 (700348334H1), SEQ ID NO: 4131 (700347763H1), SEQ ID NO: 4423 (700348396H1), SEQ ID NO: 7329 (700381547H1), SEQ ID NO: 4093 (700347688H1), SEQ ID NO: 4310 (700348209H1), SEQ ID NO: 6681 (700352229H1), SEQ ID NO: 5263 (700349865H1), SEQ ID NO: 6254 (700351489H1), SEQ ID NO: 6442 (700351811H1), SEQ ID NO: 5226 (700349807H1), SEQ ID NO: 5308 (700349935H1), SEQ ID NO: 6730 (700352306H1), SEQ ID NO: 7499 (700381835H1), SEQ ID NO: 5902 (700350873H1), SEQ ID NO: 7531 (700381889H1), SEQ ID NO: 4371 (700348314H1), SEQ ID NO: 7600 (700381994H1), SEQ ID NO: 6354 (700351650H1), SEQ ID NO: 4653 (700348801H1), SEQ ID NO: 3941 (700347405H1), SEQ ID NO: 4206 (700347960H1), SEQ ID NO: 6832 (700352474H1), SEQ ID NO: 4337 (700348259H1), SEQ ID NO: 4193 (700347929H1), SEQ ID NO: 6220 (700351433H1), SEQ ID NO: 5511 (700350274H1), SEQ ID NO: 6337 (700351625H1), SEQ ID NO: 6900 (700380811H1), SEQ ID NO: 7547 (700381923H1), SEQ ID NO: 5525 (700350293H1), SEQ ID NO: 4474 (700348485H1), SEQ ID NO: 6498 (700351910H1), SEQ ID NO: 4968 (700349359H1), SEQ ID NO: 6295 (700351554H1), SEQ ID NO: 7584 (700381973H1), SEQ ID NO: 4750 (700348978H1), SEQ ID NO: 5355 (700350018H1), SEQ ID NO: 4464 (700348467H1), SEQ ID NO: 6755 (700352345H1), SEQ ID NO: 4106 (700347718H1), SEQ ID NO: 4886 (700349211H1), SEQ ID NO: 5553 (700350334H1), SEQ ID NO: 4396 (700348356H1), SEQ ID NO: 5939 (700350936H1), SEQ ID NO: 6189 (700351371H1), SEQ ID NO: 5803 (700350709H1), SEQ ID NO: 4655 (700348803H1), SEQ ID NO: 6120 (700351265H1), SEQ ID NO: 4799 (700349054H1), SEQ ID NO: 5830 (700350748H1), SEQ ID NO: 6686 (700352236H1), SEQ ID NO: 4589 (700348678H1), SEQ ID NO: 5870 (700350819H1), SEQ ID NO: 5794 (700350692H1), SEQ ID NO: 4646 (700348782H1), SEQ ID NO: 4489 (700348513H1), SEQ ID NO: 4157 (700347830H1), SEQ ID NO: 5651 (700350478H1), SEQ ID NO: 5982 (700351019H1), SEQ ID NO: 4466 (700348472H1), SEQ ID NO: 4692 (700348872H1), SEQ ID NO: 6847 (700352506H1), SEQ ID NO: 4980 (700349375H1), SEQ ID NO: 4387 (700348343H1), SEQ ID NO: 4067 (700347631H1), SEQ ID NO: 5546 (700350324H1), SEQ ID NO: 3999 (700347509H1), SEQ ID NO: 4794 (700349046H1), SEQ ID NO: 4382 (700348333H1), SEQ ID NO: 5348 (700350008H1), SEQ ID NO: 4657 (700348806H1).
Analogous to the nucleic acid sequences, a CDP may have an amino acid sequence which is naturally occurring, synthetic, or variant. Guidance in determining which amino acid residues may be substituted, inserted, or deleted without abolishing biological or immunological activity may be found using computer programs such as LASERGENE Navigator (DNASTAR, Madison Wis.) which are well known in the art.
Derivation of Nucleic Acid Sequences
In the present embodiment, mRNA was isolated from corn ear and used to construct the SATMON022 and SATMON023 cDNA libraries. Random cDNA isolates were sequenced in part and analyzed using the homology programs described below. The sequences of the isolates are disclosed in the Sequence Listing. These cdps may contain either a partial or a full length open reading frame, or they may contain all or part of a regulatory element for a particular gene. This variation is attributed to the fact that many genes are several hundred, and sometimes several thousand, bases in length. With current technology, large genes cannot be cloned in their entirety because of vector limitations, incomplete reverse transcription of the first strand, or incomplete replication of the second strand. This is particularly common in libraries generated by random priming, since first strand synthesis may begin anywhere within the transcript. Contiguous, secondary clones containing additional nucleotide sequences may be obtained using a variety of methods known to those of skill in the art.
Sequencing of the cDNAs
Methods for DNA sequencing are well known in the art. Conventional enzymatic methods employ DNA polymerase, Klenow fragment, THERMO SEQUENASE DNA polymerase (Amersham Pharmacia Biotech, Piscataway N.J.), or Taq DNA polymerase (Amersham Pharmacia Biotech) to extend the nucleic acid sequence from an oligonucleotide primer annealed to the DNA template of interest. Methods have been developed for the use of both single-stranded and double-stranded templates. Chain termination reaction products may be electrophoresed on urea-polyacrylamide gels and detected either by autoradiography (for radionucleotide-labeled nucleotides) or by fluorescence (for fluorescent-labeled nucleotides). Recent improvements in mechanized reaction preparation, sequencing, and analysis using the fluorescent detection method have permitted expansion in the number of inserts that may be sequenced per day using machines such as the ABI 377 DNA Sequencer (Perkin Elmer, Norwalk Conn.).
Reading Frame Determinations
The reading frame of the nucleotide sequence may be ascertained by several types of analyses. First, reading frames contained within the coding sequence may be analyzed for the presence of start (ATG, GTG, etc.) and stop codons (TGA, TAA, TAG). Typically, one reading frame will continue throughout the major portion of a cDNA sequence while the other two reading frames tend to contain numerous stop codons. For more difficult cases, algorithms have been created to analyze the occurrence of individual nucleotide bases at each putative codon triplet (Fickett, J. W. (1982) Nucl. Acids Res. 10:5303-5318). Coding sequences for particular organisms (bacteria, plants, and animals) tend to contain certain triplet periodicities, such as a significant preference for pyrimidines in the third codon position. These preferences have been incorporated into widely available software which may be used to determine the coding potential and frame of a given stretch of DNA. Coding preferences and start/stop codon information may be used to determine proper frame with a high degree of certainty which, in turn, permits cloning of the sequence in the correct frame.
The nucleotide sequences of the Sequence Listing have been prepared by current, state-of-the-art, automated methods and, as such, may contain occasional sequencing errors and unidentified nucleotides. Such unidentified nucleotides are designated by an N. The infrequent sequencing errors or N""s in the nucleotide sequences of the Sequence Listing do not present a problem to those skilled in the art who wish to practice the invention. Several methods employing standard recombinant techniques, described in Ausubel, F. M. et al. (1997; Short Protocols in Molecular Biology, John Wiley and Sons, New York N.Y.), Sambrook, J. et al. (1989; Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, Plainview N.Y.), or periodic updates thereof, may be used to correct errors and complete the missing sequence information. The same techniques used for obtaining a full-length sequence may be used to obtain a complete and accurate nucleotide sequence.
Homology Searches
The nucleic acid sequences of the Sequence Listing were used as query sequences against GenBank, or other databases available to the public, to determine homology to known sequences. Illustrative of computer programs known to those of skill in the art for performing computer-assisted nucleic acid or amino acid homology searches is the program Basic Local Alignment Search Tool or BLAST (Altschul, S. F. (1993) J. Mol. Evol. 36:290-300; Altschul, et al. (1990) J. Mol. Biol. 215:403-410). BLAST produces alignments of both nucleotide and amino acid sequences to determine sequence similarity. BLAST is especially useful in determining exact matches or homology. GenBank databases may be searched for sequences containing regions of homology to a query cdp of the present invention. Other databases (such as SwissProt, BLOCKS, or Pima II) may be searched for regions of amino acid sequence homology corresponding to the deduced CDP.
As described in Karlin (supra), the fundamental unit of BLAST algorithm output is the High-scoring Segment Pair (HSP). An HSP consists of two sequence fragments of arbitrary, but equal lengths, whose alignment is locally maximal and for which the alignment score meets or exceeds a threshold or cutoff score set by the user. The parameter E establishes the statistically significant threshold for reporting database sequence matches. E is interpreted as the upper bound of the expected frequency of chance occurrence of an HSP (or set of HSPs) within the context of the entire database search. Any database sequence whose match satisfies E is reported in the BLAST program output.
BLAST may be used with any of the cdps of the present invention to search for HSPs between a query sequence and sequences in a reference nucleotide or protein database. The statistical significance of any matches is evaluated, and those matches that satisfy the user-selected threshold of significance are reported.
Homologous sequences, as determined by a BLAST search, may include prokaryotic (bacterial) or eukaryotic (animal, fungal, or plant) sequences. Where a cdp represents only part of a gene, the degree of homology is based only on the partial sequence disclosed in the Sequence Listing. When sequences have sufficiently long regions of agreement or sufficiently high overall agreement, the score of the cdp is considered to be a nearly exact match. Allelic sequences fit this category when they only differ by about three nucleotides per 100. Homologous matches between the cdps provided in the Sequence Listing and the GenBank databases are reported in TABLES 1 and 2.
Corn Ear-Derived Sequences
The cdps of the present invention may serve to identify, evaluate, alter, or follow the inheritance of desired characteristics associated with growth and development, disease resistance, environmental adaptability, quality, and yield of corn. In particular, cdps are useful as molecular markers for studying inheritance of multigene traits in a plant breeding program.
Hybridization and Genetic Analysis
The cdps, their oligonucleotides, fragments, or complementary sequences, may be used to identify the presence of and/or to determine the degree of similarity between two (or more) nucleic acid sequences. The cdps may be hybridized to naturally occurring or recombinant nucleic acid sequences under appropriately selected temperatures and salt concentrations. Hybridization with a probe based on the nucleic acid sequence of at least one of the cdps allows for the detection of nucleic acid sequences, including genomic sequences, which are identical or closely homologous to the cdps of the Sequence Listing. Probes may be selected from non-conserved or unique regions of the cdps of interest and pretested for their ability to identify or amplify the target nucleic acid sequence using standard protocols. Optimization of the protocol, e.g. increasing stringency to reduce the frequency of false positives or avoiding polyadenylated or other regions predicted to provide secondary structure to reduce false negatives, should provide the desired results. A labeled probe may be used to detect or quantify cDNAs, endogenous corn transcripts, or genes. As will be understood by those of skill in the art, hybridization conditions, probe length and labeling will vary depending upon the intended use. Hybridization conditions, based on the melting temperature (Tm) of the probe and on the salt concentrations under which hybridization and subsequent washes are carried out are well known in the art and are taught in Sambrook (supra) and Ausubel (supra).
A probe for use in Southern or northern hybridization may be a cdp sequence or its complement that is up to several hundred nucleotides long and either single-stranded or double-stranded. Such probes may be hybridized in solution to biological materials such as plasmids, bacterial or yeast artificial chromosomes, cleared plant tissues, etc. or to artificial substrates containing cdps. Microarrays are particularly suitable for identifying the presence and detecting the level of gene expression of multiple desired traits by examining gene expression of selected inbreds and is hybrids at various stages of development. An array analogous to a dot or slot blot may be used to arrange and link the cDNA fragments or oligonucleotides to the surface of a substrate using one or more of the following: mechanical (vacuum), chemical, thermal, or UV bonding procedures. Such an array may contain any number of cdps and may be produced by hand or by using available devices, materials, and machines.
Probes may be labeled by either PCR or enzymatic techniques using a variety of reporter molecules. Commercial kits are available for radioactive labeling and probe cleanup from Amersham Pharmacia Biotech, for alkaline phosphatase labeling from Life Technologies (Rockville Md.), for chemiluminescent labeling from Lumigen (Southfield Mich.), etc. Alternatively, cdps may be cloned into commercially available vectors for the production of RNA probes. Such probes may be transcribed in the presence of at least one labeled nucleotide (e.g. [xcex1xe2x88x9232P] CTP, Amersham Pharmacia Biotech).
Genetic maps, based upon molecular markers (restriction fragment length polymorphisms, RFLPs) are being assembled for several grains including rice, corn, barley, and wheat. These maps have improved understanding and manipulation of both single and multigene traits. Even when the genes involved are unknown, the ability to show the presence of the associated marker and the desired characteristics in inbred or hybrid corn plants and to follow segregation in a breeding program make the marker valuable as a diagnostic. Moreover, continuous variation within a segregating family may often be resolved into a handful of major gene effects associated with molecular markers. As genetic maps merge with physical maps, it becomes possible to walk along the chromosome and clone virtually any gene. Hybridization and newer technologies such as random amplified polymorphic DNA (RAPD) analysis, microsatellites and amplified fragment length polymorphisms (AFLP) make it easier to isolate the actual genes which interact and are responsible for a desired trait.
Diagnostic Uses
Diagnostic assays known to those of skill in the art may be used to detect or confirm conditions or diseases associated with abnormal levels of cdp expression. Labeled probes developed from the nucleotide sequence encoding a cdp are added to a plant sample under amplifying or hybridizing conditions. The complex between the naturally occurring sequence and the labeled probe is quantified and compared with a standard for that cell or tissue. If cdp expression varies significantly from the standard, the assay indicates the presence of the condition or disease. Qualitative or quantitative diagnostic methods may include northern, dot blot, or other membrane or dip-stick based technologies or multiple-sample format technologies such as PCR, ELISA-like, pin, or chip-based assays. The determination of whether cdp expression in a sample varies significantly from a standard is determined by methods of statistical analyses well known to those of skill in the art.
Accordingly, the invention provides a method for assessing disease resistance or other conditions using a panel of probes. A candidate probe is identified from CDPs which are specific to corn tissue and have not been observed in GenBank or other Incyte-sequenced cDNA libraries. The usefulness of the probe may be tested by quantifying its hybridization across tissues which are normal versus diseased. Once an increase (or decrease) in expression level is related to a trait such as fungal resistance, the probe can be used to monitor ability of a particular inbred or hybrid corn line to withstand fungal infection.
Transcript Imaging
Another embodiment relates to development of diagnostic or treatment methods based on specific imaging of the cdps of the present invention. The profile of nucleic acid sequences which reflect gene transcription activity in a particular cell type, tissue, or plant at a particular time, is defined as a xe2x80x9ctranscript imagexe2x80x9d. Such profiles are generated by naming, matching, and counting all copies of related clones and arranging them in order of abundance.
Clones may also be arranged in clusters in descending order of abundance. The minimum number of clones necessary to constitute a cluster, as illustrated at the bottom of TABLE 2, is two. All clones in TABLE 2 are ear specific although individual clusters may consist of either unique cdps or cdps that are homologous to known sequences. An alternative presentation of this data might involve a spreadsheet which contains cluster abundance data as well as some descriptive information for the homologous clones.
Subtractions, or subsetting, among transcript images may be used to discern various differences in gene expression and cellular activities. For example, subsetting may be used with the PHYTOSEQ database (Incyte Pharmaceuticals, Palo Alto Calif.) to show differences between: a) organs of two different developmental stages; b) two different organs, such as leaves and roots; c) organs from two different species; or d) normal and diseased or stressed plant tissues.
Large numbers of mRNA transcripts, as represented by their respective cDNA clones, may be compared using computational methods rather than analogous laboratory methods, such as northern blot analysis. For example, electronic subtraction between any two transcript images parallels hybrid subtraction between any two cDNA libraries (cf. Sambrook, supra). The information produced by the subtraction of transcript images between different libraries may be used to select single or multiple cdps which may be used to predict yield.
A cdp identified through transcript imaging, or other means, may also be used to clone regulatory elements for use in transformation vectors. Expression may be quantified using amplification or microarray technologies which are well known in the art.
Complementary Strand
The cdp, or any part thereof, may be used as a tool in technologies for altering gene expression. To inhibit in vivo or in vitro cdp transcription, a PNA (Nielsen, P. E. et al. (1993) Anticancer Drug Des. 8:53-63) or an oligonucleotide based on the sequence of a cdp is designed using OLIGO 4.06 software (National Biosciences, Plymouth MN) or LASERGENE Navigator (DNASTAR). Alternatively, a fragment of a cdp is cloned into an expression vector which is transformed into a host cell to express the complementary strand. An analogous molecule may be designed to inhibit promoter binding in the upstream nontranslated leader or at various sites along the 5xe2x80x2 coding region of the cdp. Alternatively, complementary molecules may be designed to inhibit translation of an mRNA by preparing an oligomer or fragment which binds to the transcript preventing its association with the ribosomal machinery.
Complementary molecules may also be designed to disrupt genomic sequences (such as enhancers, introns) preventing the normal activity of these regulatory elements. Similarly, complementary strands may be used in a process known as xe2x80x9ctriple helixxe2x80x9d base pairing to inhibit replication. These molecules compromise the ability of the double helix to open and bind to polymerases and transcription factors necessary for replication.
Stable transformation of appropriate dividing cells with an expression vector encoding the complement of a cdp may produce a transgenic cell line, tissue, or organism. Those cells which assimilate, replicate, and express the nucleic acid sequence in sufficient quantities may compromise or entirely eliminate the natural activity of the cdp. Frequently, the function of a cdp may be ascertained by observing lethality, loss of physiological activity, changes in morphology, etc. at the cellular, tissue, organ, or organismal level.
Expression
The cdps may be used in recombinant vectors to express a polypeptide. It may be advantageous to design nucleic acid sequences possessing the GC ratio of codons preferred by a particular prokaryotic or eukaryotic host (Murray, E. et al. (1989) Nuc. Acids Res. 17:477-508). In addition, 3xe2x80x2 terminators, such as bacterial nopalene synthase or octapine synthase, may be modified, or substituted into vectors, to produce transcripts having more desirable properties, such as a longer half-life, than transcripts produced from the naturally occurring sequence (Sullivan, M. L. and Green, P. J. (1993) Plant Mol. Biol. 23:1091-1104; Silva, E. M. et al. (1987) J. Cell Biol. 105:245). The cdps may also be altered by site-directed mutagenesis to insert new restriction sites and to modify the peptide by glycosylation, phosphorylation, acetylation, etc.
The cdp may be ligated to a heterologous sequence to create a chimeric or fusion protein. For ease of purification, it may be useful to produce a fusion protein that is recognized by a commercially available antibody. In addition, the sequence may be engineered to introduce a cleavage site between the peptide of interest and the heterologous protein sequence, so that the peptide may be cleaved from the heterologous moiety and purified.
Alternately, the peptide may be synthesized, whole or in part, using chemical methods well known in the art. For example, peptides may be synthesized using various solid-phase techniques (Roberge, J. Y. et al. (1995) Science 269:202-204) or an Peptide Synthesizer Model 431A (Perkin Elmer) using instructions provided by the manufacturer. Once synthesized, the peptide may be purified by preparative high performance liquid chromatography, and composition confirmed by amino acid sequencing (Ausubel (supra) p. 10.82f).
Expression Systems
For protein expression, the nucleic acid sequence may be inserted into an expression vector which contains the necessary elements for appropriate transcription and translation. Methods which are well known to those skilled in the art may be used to construct such vectors. These methods include in vitro recombinant DNA techniques, synthetic techniques, in vivo recombination, or genetic recombination. Such techniques are described in Sambrook (supra) and Ausubel (supra). One of the advantages of producing the CDPs by recombinant DNA technology is the ability to obtain highly-enriched sources of the polypeptides that simplify purification procedures.
The cdps may be engineered into a variety of expression vectors and host cells. These include, but are not limited to, microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid or cosmid DNA expression vectors; yeast transformed with yeast expression vectors; insect cell systems infected with virus expression vectors (e.g. baculovirus); plant cells transfected with expression vectors containing viral, bacterial, or eukaryotic elements (e.g. cauliflower mosaic virus, CaMV; Ti or pBR322 plasmids; and cell or ear-specific, constitutive or inducible, monocot or corn elements).
The regulatory elements of vectors- vary in their strength and specificities and are those nontranslated regions such as enhancers, promoters, introns, and 3xe2x80x2 untranslated regions which interact with host proteins to carry out transcription and translation. Depending on the vector and host, any number of suitable transcription and translation elements may be used. For example, promoters or enhancers derived from the genomes of plant cells (e.g. heat shock, RUBISCO; and storage protein genes) or from plant viruses (e.g. viral promoters or leader sequences) may be cloned into the vector containing an appropriate selectable marker. In fact, the cdps of this invention may be used to clone upstream, tissue-specific or inducible regulatory elements for purposes of engineering and expressing heterologous genes in corn.
In a bacterial system, an expression vector may be selected to direct a high level expression of a fusion protein. Commercial vectors include, but are not limited to, the multifunctional E. coli cloning and expression vectors, PBLUESCRIPT (Stratagene, La Jolla Calif.) and PSPORT (Life Technologies). Using either of these vectors, the nucleic acid sequence may be ligated into the vector in-frame with sequences for the amino-terminal Met and the subsequent 7 residues of 3-galactosidase so that a chimeric protein is produced. PGEX vectors (Amersham Pharmacia Biotech) may also be used to express peptides by ligating the nucleic acid sequence to glutathione S-transferase (GST). In general, such fusion proteins are soluble and may easily be purified from lysed cells by adsorption to glutathione-agarose beads followed by elution in the presence of free glutathione. Proteins made in such systems are designed to include heparin, thrombin, or factor XA protease cleavage sites so that the peptide of interest may be released from the GST moiety at will.
In plants, the expression of nucleic acid sequences may be driven by any of a number of promoters. Viral promoters such as the 35S and 19S promoters of CaMV may be used alone or in combination with the omega leader sequence from TMV (Takamatsu, N. et al. (1987) EMBO J. 6:307-311). Alternatively, plant promoters such as the small subunit of RUBISCO (Coruzzi, G. et al. (1984) EMBO J. 3:1671-1680; Broglie, R. et al. (1984) Science 224:838-843); or heat shock promoters (Winter, J. and Sinibaldi, R. M. (1991) Results Probl. Cell Differ. 17:85-105) may be used. Preferably, the cdps of the invention may be used to identify clones containing full length genes by hybridization or to clone full length genes or regulatory elements for use in expression vectors by PCR.
X-Ray Crystallography
Expression of the recombinant CDP in sufficient amounts may make analytical studies such as X-ray crystallography possible. In the alternative, knowledge of the amino acid sequence deduced from the nucleic acid sequence may provide guidance to those employing computer modeling techniques in place of or in addition to X-ray crystallography.
Antibodies
Anti-CDP antibodies may be produced to use in assays of protein expression. Such antibodies include, but are not limited to, polyclonal, monoclonal, chimeric, single chain, Fab fragments and fragments produced by a Fab expression library. Neutralizing antibodies, i.e., those which inhibit dimer formation, are especially useful for diagnostics.
The amino acid sequence encoded by the cdps of the Sequence Listing may be analyzed by appropriate software (e.g. LASERGENE Navigator, DNASTAR) to determine regions of high immunogenicity. The optimal sequences for immunization are selected from the C-terminus, the N-terminus, and those intervening, hydrophilic regions of the peptide which are likely to be exposed to the external environment when the peptide is in its natural conformation. Analysis used to select appropriate epitopes is also described by Ausubel (supra, unit 11-7). Peptides used for antibody induction do not need to have biological activity; however, they must be antigenic. Peptides used to induce specific antibodies may have an amino acid sequence consisting of at least five amino acids, and preferably at least 10 amino acids. An oligopeptide should mimic an antigenic portion of the natural peptide and may be fused with another protein such as KLH (Sigma-Aldrich) for antibody production. An oligopeptide or peptide encompassing an antigenic region may be expressed from the nucleic acid sequence, synthesized, or purified from corn.
Procedures well known in the art may be used for the production of antibodies. Various hosts including mice, goats, and rabbits, may be immunized by injection with a peptide or oligopeptide. Depending on the host species, various adjuvants may be used to increase immunological response.
In one procedure, oligopeptides about 15 residues in length may be synthesized using a Peptide Synthesizer Model 431A (Perkin Elmer) using Fmoc-chemistry and coupled to KLH (Sigma-Aldrich) by reaction with M-maleimidobenzoyl-N-hydroxysuccinimide ester (Ausubel, supra). If necessary, a cysteine may be introduced at the N-terminus of the oligopeptide to permit coupling to KLH. Rabbits are immunized with the oligopeptide-KLH complex in complete Freund""s adjuvant. The resulting antisera are tested for antipeptide activity by binding the peptide to plastic, blocking with 1% BSA, reacting with rabbit antisera, washing, and reacting with radioiodinated goat anti-rabbit IgG.
In another procedure, the peptide, in quantities up to 75 mg, may be used to immunize mice or rabbits. About 100 xcexcg are used to immunize a mouse, while up to 1 mg is used to immunize a rabbit. Subsequently, the peptide is radioiodinated and used to screen the B-lymphocyte cells from the immunized animal for production of hybridomas using standard techniques. About 20 mg of protein are sufficient for labeling and screening several thousand clones.
Hybridomas may also be prepared and screened using standard techniques. Hybridomas of interest are detected by screening with radioiodinated peptide to identify those fusions producing peptide-specific monoclonal antibody. In a typical protocol, wells of microtiter plates are coated with affinity-purified, specific rabbit-anti-mouse (or suitable anti-species IgG) antibodies at 10 mg/ml. The coated wells are blocked with 1% BSA and washed and exposed to supernatants from hybridomas. After incubation, the wells are exposed to radiolabeled peptide at 1 mg/ml. Clones producing antibodies bind a quantity of labeled peptide that is detectable above background.
Such clones are expanded and subjected to 2 cycles of cloning at 1 cell/3 wells. Cloned hybridomas are injected into pristane-treated mice to produce ascites, and monoclonal antibody is purified from the ascitic fluid by affinity chromatography on Protein A (Amersham Pharmacia Biotech). Monoclonal antibodies with affinities of at least 108 Mxe2x88x921, preferably 109 Mxe2x88x921 to 1010 Mxe2x88x921 or greater, are made by standard procedures as described in Harlow (1988; Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor N.Y.) and Goding (1986; Monoclonal Antibodies: Principles and Practice, Academic Press, New York N.Y.).
Antibody fragments which contain specific binding sites for an epitope may also be generated. For example, such fragments include, but are not limited to, the F(abxe2x80x2)2 fragments which may be produced by pepsin digestion of the antibody molecule and the Fab fragments which may be generated by reducing the disulfide bridges of the F(abxe2x80x2)2 fragments. Alternatively, Fab expression libraries may be constructed to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity (Huse, W. D. et al. (1989) Science 256:1275-1281).
Assays Using Antibodies
Anti-CDP antibodies may be used to determine the amount of CDP found in a particular cell of a corn inbred line or hybrid under various environmental or disease conditions. Assays for such peptides include methods utilizing the antibody and a label to detect expression in plant extracts, cells, or tissues. The peptides and antibodies of the invention may be used with or without modification. Frequently, the peptides and antibodies will be labeled by joining them, either covalently or noncovalently, with a reporter molecule.
Protocols for detecting and measuring protein expression using either polyclonal or monoclonal antibodies, are known in the art. Examples include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA) and fluorescent activated cell sorting (FACS). A two-site, monoclonal-based immunoassay utilizing monoclonal antibodies reactive to two non-interfering epitopes on a peptide is preferred, but a competitive binding assay may be employed. Such immunoassays typically involve the formation of complexes between the CDP and its specific antibody and the measurement of such complexes. These and other assays are described, among other places, in Hampton, R. et al. (1990, Serological Methods, a Laboratory Manual, APS Press, St Paul Minn.) and Maddox, D. E. et al. (1983, J. Exp. Med. 158:1211-1226).
Screening for Useful Compounds
The cdps or CDPs are particularly useful for screening libraries of molecules or test compounds for identification of those molecules which bind specifically to them. For example, a cdp or fragment thereof may be combined with a plurality of natural, synthetic, or inorganic molecules to screen for molecules that bind to them and function as transcription factors, enhancers, or other cellular elements which contribute to gene expression. Similarly, a CDP or portion thereof may be combined with a plurality of molecules to screen for molecules which bind to them. Molecules identified by screening with cdps or CDPs preferably affect growth and development, disease resistance, environmental adaptability, quality, or yield.
Technologies with multiple-sample format, ELISA-like, capillary, or chip-based assays, are well known in the art and allow large-scale screening. These methods use complex formation, quantification, and comparison with a standard to detect molecules which specifically bind the cdps or CDPs. In the assay, the cdp or CDP may be free in solution, affixed to a substrate, borne on a cell surface, or located intracellularly. For example, prokaryotic host cells which are stably transformed with recombinant nucleic acids that express and position a CDP on the cell surface can be used to screen for molecules which specifically bind the CDP. Viable or fixed cells are screened against a plurality of test compounds and the specificity of binding or formation of complexes between an expressed CDP and the test compound is measured.
Transformation
Bacterial and plant transformation systems are well known in the art. Expression vectors may be introduced into suitable E. coli cells by electroporation, heat shock or other means as described in Ausubel (supra) for the purpose of expressing a plant protein. Expression vectors may also be introduced into plant cells by direct transfer of DNA or pathogen-mediated transfection. For reviews, see McGraw Hill Yearbook of Science and Technology (1992; McGraw Hill New York N.Y., pp 191-196); or Weissbach, A. and Weissbach, H. (1988; Methods Enzymol. 118:421-463). Direct transfer of DNA into plant protoplasts or cells is one approach for transforming plants genetically. DNA uptake by protoplasts may be promoted chemically with polyethylene glycol or electrically with a high-voltage pulse. Both of these methods depend upon a cell culture system to recover plants from a single transformed cell (Rhodes, C. A. et al. (1988) Biotechnology 6:56-60; Morocz, S. (1991) Theor. Appl. Genet. 80:721-726). Regeneration of transformed, fertile plants has been demonstrated in several cereals including rice (Zhang, H. M. (1988) Plant Cell Rep. 7:379-384).
Electroporation, lipofection, microinjection, particle bombardment, vacuum infiltration, and electrotransformation may be used to transform corn cells and embryos. Gordon-Kamm, W. J. et al. (1992; Plant Mol. Biol. 18:201-210) used particle bombardment to transform embryogenic, suspension culture cells; Murry, L. E. et al. (In: Bajaj, Y. P. S. (1994) Biotechnology in Agriculture and Forestry 25:252-261) used continuous, low voltage electric current to transform embryos; and Rhodes, C. A. et al. (1995; Methods Mol. Biol. 55:121-131) describe the electroporation of embryos. Stable transformation requires the use of an expression vector which contains an appropriate origin of replication and gene cassettes containing viral or plant expression elements, a selectable or visible marker, and a gene of interest. Following the introduction of the vector, cells may be allowed to grow for 1-2 days in an enriched media. If the vector contains a selectable marker, the cells are switched to selective media. The selectable marker confers resistance to selective agents and allows growth and recovery of those cells which successfully express the introduced sequences.
Any number of selection systems may be used to recover transformed cell lines. Antimetabolite, antibiotic or herbicide resistance may be used as the basis for selection using genes such as dhfr, which confers resistance to methotrexate (wigler, M. et al. (1980) Proc. Natl. Acad. Sci. 77:3567-3570); npt, which confers resistance to the aminoglycosides, neomycin and G-418 (Colbere-Garapin, F. et al. (1981) J. Mol. Biol. 150:1-14); and als or pat, which confer resistance to chlorsulfuron and phosphinotricin acetyltransferase, respectively (McGraw Hill Yearbook of Science and Technology, supra). Recently, the use of visible markers has gained. popularity with such markers as anthocyanins, xcex2 glucuronidase and its substrate, GUS, luciferase and its substrate, luciferin, and green fluorescent protein, GFP, being widely used not only to identify transformants, but also to quantify the amount of transient or stable protein expression attributable to a specific vector system (Rhodes (1995) supra; Haseloff, J. and Amos, B. (1995) Trends Genet. 11:328-329). Plant expression vectors contain 5xe2x80x2 promoters, enhancers and 3xe2x80x2 terminators that will function in the plant cell.
Identification of Transformants
Although the presence/absence of marker gene expression suggests that the gene of interest is also present, its expression should be confirmed. For example, if the sequence is inserted within a marker gene sequence, cells containing the recombinant sequence may be identified by the absence of marker gene function. Alternatively, a marker gene may be placed in tandem with the nucleic acid sequence under the control of a single promoter. Expression of the marker gene in response to induction or selection may indicate the presence and expression of the tandem sequence as well.
Alternatively, host cells which contain the introduced nucleic acid sequence may be identified by a variety of procedures known to those of skill in the art. These procedures include, but are not limited to, DNA-DNA or DNA-RNA hybridization and protein bioassay or immunoassay techniques which include membrane, solution, gel, or chip based technologies for the detection and/or quantification of the nucleic or amino acids and any of the molecules to which they bind.
The presence of the nucleic acid sequence may be detected by DNA-DNA or DNA-RNA hybridization or amplification using probes comprising all or a portion of a nucleic acid sequence. Nucleic acid amplification based assays involve the use of oligonucleotides or oligomers based on the nucleic acid sequence to detect transformants containing the introduced DNA.
A wide variety of labels and conjugation techniques are known by those skilled in the art and may be used in various nucleic acid and amino acid assays. Means for producing labeled hybridization or PCR probes for detecting related sequences include oligolabeling, nick translation, end-labeling, or PCR amplification and are well known in the art. Alternatively, the nucleic acid sequence may be cloned into a vector for the production of an mRNA probe. Such vectors are known in the art, are commercially available, and may be used to synthesize RNA probes in vitro by addition of an appropriate RNA polymerase such as T7, T3, or SP6 and labeled nucleotides. A number of companies (e.g., Amersham Pharmacia Biotech and Life Technologies) supply commercial kits, reporter molecules, and protocols for these procedures.